MIR169 have remained unknown. In this study, we show that Elongated Hypocotyl 5-Homolog (HYH) directly binds to the promoter of MIR169a and negatively regulates its expression. Absolute quantification identified MIR169a as the major locus producing miR169. GUS reporter assays revealed that the deletion of a 498-bp fragment  of MIR169a abolished its ambient temperature-responsive expression. DNA-affinity chromatography followed by liquid chromatography-mass spectrometry analysis identified transcription factor HYH as a trans-acting factor that binds to the 498-bp promoter fragment of pri-miR169a. Electrophoretic mobility shift assays and chromatin immunoprecipitation–quantitative PCR demonstrated that the HYH.2 protein, a predominant isoform of HYH, directly associated with a G-box-like motif in the 498-bp fragment of pri-miR169a. Higher enrichment of HYH.2 protein on the promoter region of MIR169a was seen at 23°C, consistent with the presence of more HYH.2 protein in the cell at the temperature. Transcript levels of pri-miR169a increased in hyh mutants and decreased in transgenic plants overexpressing HYH. Consistent with the negative regulation of MIR169a by HYH, the diurnal levels of HYH mRNA and pri-miR169a showed opposite patterns. Taken together, our results suggest that HYH is a transcription factor that binds to a G-box-like motif in the MIR169a promoter and negatively regulates ambient temperature-responsive expression of MIR169a at higher temperatures in Arabidopsis.Arabidopsis microRNA169 (miR169) is an ambient temperature-responsive microRNA that plays an important role in stress responses and the floral transition. However, the transcription factors that regulate the expression of  Arabidopsis thaliana (Arabidopsis), exposure to a prolonged period of low temperature  can shorten the time of flowering, especially in winter accessions. Small changes in temperature within the tolerable range, i.e., ambient temperature, also affect physiology and development of numerous plant species. For example, fluctuation of ambient temperature correlates with an increase of respiration rates  can induce sequence-specific gene silencing transcriptionally and post-transcriptionally , and regNuclear Factor Y Subunit A (NF-YA) family genes  and the bZIP transcription factor domain in the C-terminal half  (Elongated Hypocotyl 5 (HY5) and its homolog HY5-Homolog (HYH) are basic leucine zipper (bZIP) transcription factors that function in lateral root formation, secondary root thickening, and photomorphogenesis of seedlings . In A. tnal half . Due to nal half  and GATAnal half . In addind EXP8) .MIR169 itself remains unexplored. In this study, we identified cis-acting elements and a transcription factor involved in the regulation of ambient temperature-responsive expression of MIR169. We found that HYH binds the region of the MIR169 promoter required for response to ambient temperature and represses MIR169 expression. Taking our results together, we propose that HYH acts in the ambient temperature pathway as a transcription factor that binds to a G-box-like motif upon increasing temperature to repress the expression of MIR169a.Although several reports have examined miR169-mediated regulation of target gene expression , the upsArabidopsis thaliana (L.) ecotypes Columbia (Col-0) and Wassilewskija-2 (Ws-2) were used in this study. The hyh mutants, which are in the Ws-2 background, were generously provided by Prof. Gareth Jenkins . MIM169, a target mimic of miR169 that was designed to target miR169a, b, c, h, i, j, k, l, m, and n, was used to inhibit the miR169 activity (-2 s-1. Eight-day-old seedlings (unless otherwise indicated) were sampled and immediately frozen in liquid nitrogen prior to RNA or protein extraction.activity . Plants MIR169a along with a series of 5′-deletions of the promoter  were amplified from the genomic DNA using PCR. Each amplicon was named p169.1, p169.2, p169.3, p169.4, and p169.5, respectively. The full-length promoter and 5′-deletion fragments were then cloned into the pBI101 vector , which contains the β-glucuronidase (GUS) reporter gene. We named the resulting constructs p169.1::GUS, p169.2::GUS, p169.3::GUS, p169.4::GUS, and p169.5::GUS. To generate MIR169a-overexpressing plants, the genomic fragment of MIR169a was amplified by PCR, and then cloned into the pCHF3 binary vector harboring the 35S promoter. To generate HYH-overexpressing plants, HYH cDNAs were amplified from total RNA using RT-PCR and then cloned into the pBA-HA binary vector. The sequence of each plasmid construct was verified by sequencing . The constructed plasmids were then introduced into wild-type Col-0 plants using the floral dip method  of p method . The hom kb of p PP2AA3 (AT1G13320) and a SAND family gene (AT2G28390)  data for Col-0 plants grown at 23°C (GSE87851) . The extracted RNA was treated with DNase I  to eliminate DNA contamination, then cDNA was synthesized using the Transcriptor First-Strand cDNA Synthesis Kit . The transcript levels were measured by quantitative real-time PCR (qRT-PCR), using Green I Master Mix  with specific primers (Supplementary Table 2G28390) . All rea2G28390) , using sSE87851)  were dowFor western blot experiments, total proteins were extracted with Pro-Prep protein extraction buffer (Intron Biotechnology) using 8-day-old seedlings grown on MS media supplemented with 1.5% sucrose at 16 and 23°C under LD conditions. Samples were harvested at zeitgeber time 4 (ZT4). HA:HYH protein was detected using monoclonal anti-HA-antibodies  and polyclonal anti-Actin-antibodies (Agrisera) were used to detect actin, which was used as a loading control.GUS under the control of the MIR169a promoter fragments. The samples were incubated for 12 h at 37°C with the substrate solution . Stained seedlings were washed with 70% ethanol to eliminate chlorophyll, and were then photographed with a Nikon SMZ1000 Stereomicroscope . GUS enzymatic activity was quantified using the GUS Fluorescent Reporter Gene Activity Detection Kit  according to the manufacturer’s instructions. The assay was performed with three biological replicates, each with three technical replicates. Statistical analysis for the enzymatic activity was conducted using one-way analysis of variance (ANOVA), and the means were compared using Duncan’s Multiple Range Test.Histochemical localization of GUS was analyzed in the 8-day-old transgenic seedlings expressing MIR169a promoter, and generated from the genomic DNA using PCR with one 5′-biotin-modified and one unmodified primers  was cloned from cDNA using RT-PCR , sonicated, then centrifuged. The supernatant containing soluble HYH.2-His protein was collected and then purified using His GraviTrap . SDS–PAGE and western blotting using anti-His antibody were then carried out to check the protein quality and identity, respectively.To produce His-tagged HYH protein, the open-reading frame of Arabidopsis The DNA sequences of the predicted HYH-binding motifs were synthesized with a 5′-biotin label . Electrophoretic mobility shift assays (EMSAs) were performed using the LightShift Chemiluminescent EMSA Kit  according to the manufacturer’s instructions. Double-stranded 5′-biotin-labeled oligonucleotides were used as DNA probes (Supplementary Table Chromatin immunoprecipitation (ChIP) was performed as described  using 12A. thaliana genome, comprising the largest family among Arabidopsis miRNAs. To identify the major locus of the miR169 family, we performed absolute quantification using qPCR to determine the abundance of transcripts from each pri-miR169 locus from wild-type Arabidopsis plants at 23 and 16°C. As shown in Figure 1, pri-miR169a was the most abundant of all transcripts generated from the loci , followed by pri-miR169m, l, k, and d, which had similar amounts of transcripts . By contrast, pri-miR169g, f, c, i, b, and j showed very low levels of transcripts . The results suggested that MIR169a was the major locus of the miR169 family at both 23 and 16°C and furthermore its expression levels were approximately four times higher at 16 than at 23°C. The 14 loci produce four mature miR169 isoforms; therefore, we suggest that the miR169a isoform has the highest abundance, followed by miR169h–n, d–g, and bc. Consistent with our observation, deep-sequencing results also suggest that MIR169a is the major locus of the miR169 family  by PCR and then cloned into the promoter-less binary vector pBI101 containing a GUS reporter gene, and named p169.1::GUS. In addition, promoter deletion fragments with serial deletions of approximately 500 bp from the 5′-end were generated using PCR, which resulted in promoter fragments of 1,668, 1,170, 590, and 141 bp . These fragments were cloned into the promoter-less binary vector pBI101 to generate p169.2::GUS, p169.3::GUS, p169.4::GUS, and p169.5::GUS constructs, respectively. All constructs were introduced into wild-type Arabidopsis Col-0 using the Agrobacterium-mediated floral dip method. The transformants were screened on MS media containing kanamycin, and then checked for the presence of the transgene insertion by PCR (data not shown). Homozygous lines were isolated from the T3 generation and used for subsequent experiments.The full-length promoter 2,149 bp) of 49 bp of MIR169a promoter, we first performed fluorometric GUS assays using transgenic plants containing the different MIR169a promoter fragments. At 23°C, GUS activities of plants harboring p169.1, p169.2, and p169.3 constructs were high but their levels were not statistically different. However, at 16°C, the highest levels of GUS activity were observed in the transgenic plants harboring p169.1 and p169.2 construct, and the activity was significantly induced at 16°C by approximately 2.7-fold, consistent with a previous study that reported higher accumulation of mature miR169 at 16°C  was crucial for the ambient temperature response, raising a possibility that the regulatory elements are present in the 498-bp fragment. Further deletion of promoter to the -427 position (p169.4::GUS) caused a dramatic loss of promoter activity at both temperatures, indicating that the core promoter of the MIR169a gene is located between the -1,007 and -427 positions. GUS activity was completely abolished in the +22 deletion (p169.5::GUS). The pBI101 transgenic line, which contains a promoter-less GUS gene, showed no induction at both temperatures, as seen in p169.5::GUS plants.To examine the ambient temperature inducibility and identify the ambient temperature-responsive region of the  at 16°C . Since tMIR169a promoter activity, the homozygous pMIR169::GUS plants were subjected to histochemical GUS staining. As expected, strong GUS staining was observed in p169.1::GUS plants at 16°C. Strong staining was detected in the cotyledons of the seedlings grown at 16°C. The hypocotyl, root, and true leaves also showed weaker GUS staining. Similar staining patterns were observed at 23°C but to a comparatively lesser extent . In p169.2::GUS seedlings, the staining was still strongly detected in the cotyledon and hypocotyl, but not in the root. This implies that the region between -1,986 and -1,505 of the promoter contains a sequence that confers root-specific expression. The overall GUS staining intensity was higher at 16°C than at 23°C in p169.2::GUS plants, consistent with the results from the enzymatic activity assays. Notably, no apparent difference in GUS staining intensity was observed in p169.3::GUS plants at the two different temperatures. The p169.4::GUS and p169.5::GUS plants showed no visible GUS staining. The pBI101 plants, which contain the promoter-less GUS gene, did not show any GUS staining. These results suggested that the expression of MIR169a is upregulated by a low ambient temperature at the transcriptional levels and a cis-acting element that responds to ambient temperature changes is located in the 498-bp fragment  of the MIR169a promoter.To investigate the spatial and tissue-specificity of MIR169a expression; we therefore postulated that transcription factors involved in the ambient temperature response might bind to this region and regulate MIR169a expression. To identify such transcription factor(s), DNA-affinity chromatography was performed, using the 498-bp fragment as a DNA bait, and total protein extracted from 8-day-old seedlings grown at 16 and 23°C. We eluted affinity-captured proteins and visualized them after SDS-PAGE. After silver staining, we identified a single band (about 75 kDa) at 23°C , but not at 16°C, suggesting that the eluted proteins bind to the 498-bp fragment of the MIR169a promoter in planta. The control reactions that used the same protein extracts without the 498-bp fragment as a bait did not elute these proteins.The deletion analysis showed that the 498-bp fragment is important for ambient temperature-responsive Figure 4A was excised from the gel and then subjected to LC-MS to identify its amino acid composition. Twelve peptide signature sequences were recovered by LC-MS. By comparing them against the Arabidopsis protein database using Protein BLAST, four peptide sequences were identified as being encoded by the ORF of AT3G17609, which encodes the bZIP transcription factor protein Elongated Hypocotyl 5-Homolog (HYH) . The remaining eight peptide sequences belonged to the ribosomal protein L22p/L17e family protein encoded by AT1G52370 . As the ribosomal protein is thought to be ubiquitous in the cytosol, we regarded the ribosomal protein as a contaminant. These results suggest that HYH protein directly interacts with the cis-acting element(s) that are present within the 498-bp fragment  of the MIR169a promoter to regulate MIR169a expression at 23°C.To identify the unknown proteins, the band in HYH , consistent with a previous report , we identified four splicing variants (HYH.1–HYH.4) after sequencing. This was due to the fact that amplicons from HYH.1 and HYH.4 were only 3-nt different in size . Amino acid alignment showed that of the four variants, HYH.2 is the longest, followed by HYH.1, which contains an extra codon at the 3′-splicing site, compared to HYH.4 . HYH.3 appeared to be the shortest. Although all four HYH isoforms harbor the bZIP DNA-binding domain , only HYH.2 contains the Constitutive Photomorphogenic1 (COP1) interaction domain, which was previously suggested to have a non-canonical interaction with COP1 , respectively] . To validate the RNA-seq results, absolute quantification was performed to quantify and compare the transcript abundance of HYH.2 and HYH.3 splicing variants in 8-day-old wild-type plants at 23°C. The results showed that HYH.2 had ∼13,000 transcripts/ng of total RNA, while HYH.3 had ∼4,000 transcripts/ng of total RNA , implying that HYH.2 is the most abundant HYH splicing variant. Therefore, we chose HYH.2 for the subsequent experiments.Our RNA-seq data  suggestein silico analyses, which identified four potential HYH-binding motifs within the 498-bp fragment of the MIR169a promoter: one A-box . The sequences of the identified motifs and their locations relative to the major TSS  that were bound by HYH, we conducted ne A-box , two GATne A-box , and onene A-box   in the promoter of MIR169a, recombinant His-tagged full-length soluble HYH.2 protein (approximately 20 kDa) was produced and purified on histidine affinity columns. The purified protein displayed a single major band after elution from the column at the expected molecular weight . We performed an immunoblot analysis using anti-His antibody to confirm that the purified product contained HYH.2-His protein. Anti-His antibody successfully detected HYH.2-His protein at the expected size after blotting .To investigate whether HYH binds to the Figure 6B) were allowed to interact before being separated by electrophoresis. The results showed that HYH.2-His bound to the G-box-like motif, as a shifted band was observed for the G-box-like DNA probe. This suggested that HYH binds to the G-box-like motif within the 498-bp fragment of the promoter of MIR169a. In contrast, the A-box, both GATA boxes, as well as the negative control (NC1) did not show detectable shifted bands . To test whether the G-box-like sequence was accountable for the interaction, the core of the motif was mutated with G to T, or A to C . The binding reaction between HYH.2-His and mG failed to produce any apparent band-shifts , suggesting that HYH specifically binds to the G-box-like motif in the 498-bp fragment. To further confirm the binding affinity, unlabeled competitor probe of the identical sequence was used to perform the competition assay. The results showed that the unlabeled probe was able to compete for HYH protein binding to the G-box-like motif, as the intensity of the shifted bands gradually decreased with the increasing concentrations of unlabeled probe .To confirm that HYH protein indeed bound to the DNA motif(s) and was responsible for the interaction with the promoter, an EMSA was performed. Purified HYH.2-His protein and synthesized double-stranded 5′-biotin-labeled oligonucleotides of the possible HYH-binding motifs . A region downstream of the hairpin loop structure of miR169 was also used as a negative control (NC2) for ChIP–qPCR experiments . Taken together, our results suggested that HYH protein directly binds to the G-box-like motif of MIR169a in vitro and in vivo, especially at 23°C, to regulate its transcription.To further confirm the association of HYH with the HYH and MIR169a, pri-miR169a expression levels were measured using qPCR in hyh mutants grown at 16 and 23°C. The results showed that the levels of pri-miR169a were upregulated in hyh mutants at both temperatures when compared to the wild-type Ws plants , indicating that MIR169a expression is induced upon the depletion of HYH. We also observed an increase of pri-miR169a transcript levels at 16°C in wild-type Ws-2 plants , which is consistent with a previous report of higher accumulation of mature miR169a in wild-type Col-0 plants at 16°C . Moreover, pri-miR169a levels were less responsive to the ambient temperature changes in 35S::HA:HYH plants. At 23°C, the suppression of pri-miR169a by overexpression of HYH was not dramatic, probably because the pri-miR169a levels were already low at a high ambient temperature (23°C). To further investigate, we measured the levels of HYH transcripts in the 35S::MIR169a plants and 35S::MIM169 plants, in which the cleavage activity of most miR169 isoforms is inhibited , or inhibition of miR169 function  at both temperatures, indicating that alteration of miR169 activity did not affect the transcript levels of HYH. This suggested that HYH did not act downstream of miR169a. The expression levels of HYH in Col-0 plants at both temperatures were similar, suggesting that the ambient temperature does not regulate HYH at the transcriptional level.We measured the levels of nhibited . Our resNF-YA genes , suggesting that NF-YA1 might be the target gene of miR169a. However, as it is proposed that 35S::MIM169 plants block the action of three miR169 isoforms  . Since we observed a consistent relationship between expression of the negative regulator and the target gene of miR169, this result suggested that upregulation of NF-YA1 was due to the depletion of miR169a by the overexpression of HYH.Mature miR169 consists of four isoforms produced from 14 loci , and eacYA genes . To idenot defg) , the datce versa . To evalHYH and pri-miR169a transcripts. We sampled 7- and 8-day-old wild-type Col-0 seedlings every 4 h from ZT0 to ZT44. Consistent with the previous report of induction of HYH expression by light . The levels of pri-miR169a showed an opposite pattern, remaining low during the light period, starting to increase at ZT16, and peaking at ZT24, just before the beginning of the next light period, when HYH transcription would be induced. Our results demonstrated a clear opposite relationship between the diurnal oscillation patterns of HYH and pri-miR169a transcripts, consistent with the negative role of HYH in the regulation of MIR169a transcription.We then analyzed the diurnal rhythm of by light , HYH traMIR169a, an ambient temperature-responsive miRNA gene . The DNA-affinity chromatography coupled with LC-MS showed that HYH bound to the -1,505/-1,007 promoter region . EMSA data showed that HYH protein directly bound to a derivative of G-box motif, which is positioned at -1,167/-1,157 in the promoter . Mutation of the core G-box-like sequence prevented the interaction with HYH , supporting the idea that the G-box-like motif is crucial for the interaction between HYH and MIR169a. The genetic interaction using hyh mutants and plants overexpressing HYH, MIR169a, and MIM169 revealed that at a higher ambient temperature, HYH protein acted upstream of MIR169a as a negative regulator . Our finding on the negative role of HYH in the regulation of MIR169a expression is consistent with the observation that hyh mutants flower early . This suggested that ambient temperature might regulate HYH levels post-transcriptionally. Interestingly, COP1, an E3 ubiquitin ligase that is known to promote degradation of HYH and HY5  to demonstrate the potential upstream negative regulation of MIR169a by ambient temperature via HYH protein. Upon exposure to a higher ambient temperature, HYH binds to the G-box-like motif in the promoter of MIR169a to repress its expression and subsequently decrease the abundance of miR169 at higher temperatures. At a lower temperature, HYH fails to bind to the MIR169a promoter; thus, MIR169a transcription occurs, which leads to more pre-miR169a and mature miR169a production. Since HYH is known to be involved in photomorphogenesis and light signaling . The G-box motif is a cis-acting element that regulates responses to UV irradiation, anaerobiosis, abscisic acid, and light , and the mutation of the core motif failed to interact with HYH protein , we conclude that the G-box-like motif is required for the ambient temperature response of the MIR169a promoter.The expression of nd light . Severalnd light . Since tcis-acting elements involved in high and low temperature perception mechanisms include Abscisic Acid Response Element (ABRE), C-repeat/DRE (cis-acting element conferring ambient temperature responsiveness. Considering that the G-box is a hexameric sequence (5′-CACGTG-3′), a derivative of the G-box identified in our study, despite its sequence similarity to the G-box, could possibly be an entirely different type of cis-acting element. The sequences flanking the core G-box affect the specificity of protein binding . Moreover, the opposite diurnal shifts observed in MIR169a and HYH expression levels either resulted from HYH depletion  or light signals that might directly regulate MIR169a expression due to the presence of multiple light-responsive cis-acting elements in its promoter . These observations support the notion that the ambient temperature regulation of MIR169a expression via the HYH might be modulated by crosstalk with the light-sensing mechanism.Temperature and light are environmental cues recognized by plants as indicators of seasonal changes. Therefore, the mechanisms that perceive and integrate temperature and light signals are crucial for plant survival. There is an increasing body of evidence suggesting the existence of crosstalk between the ambient temperature and light-sensing mechanism in Arabidopsis; for instance, emerging evidence shows that phytochromes act as temperature sensors . Previouby light , our finUnderstanding how plants response to fluctuating temperature at the molecular level is a crucial step that must be taken to predict plant responses to climate change, mitigate the damage to agriculture, and support the conservation of biodiversity. Without activating temperature stress responses, ambient temperature influences plant growth and development , diseasePS designed, performed, and analyzed data from the experiments. HS performed the experiments and data analysis. JA designed and supervised the study. PS, HS, and JA wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasma cell leukemia is a rare and aggressive plasma cell neoplasm that may either originate de novo (primary PCL) or by leukemic transformation of multiple myeloma (MM) to secondary PCL (sPCL). The prognosis of sPCL is very poor, and currently no standard treatment is available due to lack of prospective clinical studies. In an attempt to elucidate factors contributing to transformation, we have performed super-SILAC quantitative proteome profiling of malignant plasma cells collected from the same patient at both the MM and sPCL stages of the disease. 795 proteins were found to be differentially expressed in the MM and sPCL samples. Gene ontology analysis indicated a metabolic shift towards aerobic glycolysis in sPCL as well as marked down-regulation of enzymes involved in glycan synthesis, potentially mediating altered glycosylation of surface receptors. There was no significant change in overall genomic 5-methylcytosine or 5-hydroxymethylcytosine at the two stages, indicating that epigenetic dysregulation was not a major driver of transformation to sPCL. The present study constitutes the first attempt to provide a comprehensive map of the altered protein expression profile accompanying transformation of MM to sPCL in a single patient, identifying several candidate proteins that can be targeted by currently available small molecule drugs. Our dataset furthermore constitutes a reference dataset for further proteomic analysis of sPCL transformation. It can entities . The oveentities  and of tentities . sPCL isentities . Primarys [CCND1 , CCND3 [1 [CCND3 , MMSET/FET/FGFR3 , cMAF [9AF [MAFB . Examplend K-RAS , deletiond K-RAS  and Rb [nd K-RAS . Interesnd K-RAS , 15, sugnd K-RAS . Immunopnd K-RAS  and CD56nd K-RAS . Finallyckpoints .13C614N2-lysine and 13C614N4-arginine are utilized, which produce a mass difference of 8.0142 Da and 10.00827 Da, respectively, for each tryptic peptide). The SILAC approach is mainly limited to proliferating cells that can be metabolically labeled with heavy amino acids. For studying e.g. patient samples, this can be circumvented by using a heavy-labelled protein mixture as internal standard. In this approach, denoted super-SILAC, a mixture of proteins extracted from several SILAC-labeled cell lines serve together as the spike-in standard. By spiking the same amount of SILAC-labeled standard in each sample, a precise relative comparison of protein levels between samples can be indirectly obtained. Super-SILAC holds tremendous potential in e.g. clinical diagnosis. This was demonstrated by Deeb et al [Although the above studies have provided some clues towards understanding factors driving the progression from MM to sPCL, to the best of our knowledge no attempts have been made to study alterations at the whole-proteome level accompanying the transformation. Such studies are by no means trivial. sPCL is very rare, thus sufficient samples for robust statistical evaluation will be extremely hard to obtain. Moreover, by escaping the bone marrow into peripheral blood the malignant plasma cells will likely adapt to the novel environment by modulating expression of several proteins. Deciphering drivers and bystanders may thus be a challenging task. To initially address these questions we here present a super-SILAC  quantitaeb et al , who werIGH-translocations. However, nearly all available MM cell lines are derived from non-hyperdiploid myelomas, and would thus not fully represent the disease [To accurately quantify proteome differences between multiple myeloma and sPCL, we generated a super-SILAC cell library consisting of three non-hyperdiploid  and three hyperdiploid  MM cell lines, as well as two B-cell lymphoma cell lines . Importantly, about 55-60% of multiple myelomas are hyperdiploid with 48-74 chromosomes. They often contain trisomies of the odd numbered chromosomes 3, 5, 7, 9, 11, 15, 19, and 21 and less frequently  disease , 24. We  disease , KJON an disease , and whi disease . Finally disease . The celTo further characterize the cell lines we prepared duplicate protein extracts from each of their non-labelled versions and individually spiked these 1:1 with the super-SILAC mixture. Each of the combined extracts was then analyzed by LC-MS/MS in at least two technical replicates as described in Materials and Methods. Joint analysis of the result files in MaxQuant identified a total of ~5270 different protein groups from the cell lines (on average ~3100 per cell line). To compare the cell line proteomes we quantified all measurements against each other based on the ratios to the super-SILAC mix and calculated their correlation coefficients. Further, we performed unsupervised hierarchical clustering of the quantified proteins using normalized SILAC-ratios to reveal the degree of pairwise differences in protein expression patterns at a global level Figure . In each2 transformed in order to have a better approximation to normal distribution. Further, median of technical replicates were calculated to represent the samples in order to be less sensitive to outliers. These are presented in In our super-SILAC analysis of MM and sPCL samples an average of 3659 ± 396 (SD) proteins were identified, amongst which 2784 ± 249 were quantified by super-SILAC. To extract as much information as possible from the RAW data, label-free quantification (LFQ) light channel from super-SILAC representing the sample were also used for quantitative analysis , 30. Pro2 super-SILAC ratios between the quantifications in MM and sPCL is shown in Figure 2 cut-off of 0.58 and p < 0.05  between the sPCL and MM samples. To analyze the subcellular location of the differentially expressed proteins, IDs Gene Ontology (GO) database annotations were analyzed. Here, 36.0 % of the proteins were reported to be localized to the cytosol, 30.6% to the nucleus and 16.2% to the mitochondria. Annotation enrichments analysis [2 ~ 0.61 p-value < 2E-11 with FDR < 5E-9), while mitochondrial proteins were down-regulated  in the sPCL cells, indicating that the transformation was accompanied by increased nuclear and reduced mitochondrial function in general. Interestingly, reduced mitochondrial function and an increased glycolytic phenotype are often observed in tumor cells even when oxygen is present. This is commonly known as the “Warburg effect” [Missing values were imputed following the guidelines from Deeb et al.  but withanalysis  over the effect”  and is a effect” . A shift effect” .−10) where 9 out of 14 associated proteins were found to be differentially expressed and seven of which were down-regulated in PCL. Colanic acid is an extracellular bacterial polysaccharide also known as M-antigen. In humans, the nucleotide sugar building blocks are instead used in other pathways such as the glycosylation of cell surface receptors. Manual inspection of the data indeed revealed that many of the enzymes involved in the formation of nucleotide-activated sugars for glycoprotein synthesis, including of GDP-mannose, GDP-fucose, UDP-N-acetylglucosamine (UDP-GlcNAc) and CMP-N-acetylneuraminic acid (CMP-Neu5Ac) were significantly down-regulated at the sPCL stage. In the GDP-mannose synthetic pathway PMM2, GMPPA and GMPPB were 1.75-, 1.92- and 2.13-fold down-regulated (p < 0.05), respectively (N-acetylneuraminic acid (Neu5Ac) in humans. To our knowledge, only one study has addressed the potential involvement of altered sialylation in MM cell trafficking [in vivo, thus establishing a role of sialylation in MM cell trafficking. Conversely, reduced α-2,3-sialylation was recently reported in colorectal tumors compared to normal tissues, indicating that the impact of sialylation is cancer-specific. Unfortunately, ST3GAL6 was not quantified in our dataset. However, the α-2,6-sialyltransferase ST6GAL1 was 1.68-fold (p~0.001) down-regulated. Together with the reduced levels of enzymes involved in sialylation precursor synthesis, this strongly suggest that α-2,6-sialylation is reduced in the sPCL samples compared to the MM samples. The precise contribution of this as well as to the formation of other glycoconjugates to the disease progression in general must, however await further studies.To search for modified biological pathways associated with transformation to sPCL, gene identifiers in the SILAC dataset were mapped in the Ingenuity® Knowledge Base (IPA) and plotted onto their canonical pathways. The ten most significant pathways are presented in Figure ectively . In the fficking . Here, hAfter their affinity maturation in germinal centers, plasma cells home to mucosal surfaces, sites of inflammation as well as to the bone marrow. This is generally accompanied by down-regulation of lymphoid tissue receptors and up-regulation of receptors for chemokines produced at the different sites. In the bone marrow, vascular cell-adhesion molecule 1 (VCAM-1) is an important mediator of plasma cell homing, through interaction with the integrin VLA-4 (ITGA4/ITGB1 dimer). Moreover, plasma cells express surface CXCR4 that migrates towards CXCL12 produced by bone marrow stromal cells as well as SDC1  that binds to fibronectin and collagen, and the adhesion molecule CD44. Intuitively, it would be expected that evasion of the malignant myeloma cells from the bone marrow is promoted by down-regulation of these surface proteins. However, no significant difference in SDC1-expression was observed in the MM and sPCL cells, whereas CD44 was 2.3-fold up-regulated (p~4.3E-05). Variants of this protein (CD44v8-v10) apparently affects the endothelial cells of small blood vessels by promoting phosphorylation of vascular endothelial cadherin, junction disruption and transendothelial migration of melanoma cells . RecentlThe second most affected pathway in IPA was TCA cycle (p~1.7E-06), in which 8 out of 23 proteins  were differentially expressed, all of which were significantly down-regulated Figure . This is+ to drive glycolytic ATP production. Targeting of glycolytic enzymes has emerged as an attractive strategy to selectively kill tumor cells, and several drugs are now in various phases of clinical trials [+ synthesis inhibitor FK866 [Down-regulation of mitochondrial respiration is commonly observed in tumor cells, with a concomitant increase in glycolytic metabolism. The Warburg effect was originally thought to be an adaption of cancer cells to the low oxygen tension in rapidly growing tumors . Howeverl trials . Blockinl trials , 52. Inhl trials . Here, tl trials  and has l trials . Finallyor FK866 .per se. There is a significantly higher mean oxygen tension in the peripheral blood (12%) compared to bone marrow (6.6%) [It is not likely that the apparent metabolic switch in the malignant cells at the sPCL stage compared to the MM stage is mediated by the change of microenvironment w (6.6%) , which sw (6.6%)  and refeIn addition to the affected pathways as revealed by IPA analysis, several other proteins were found to be differentially expressed that potentially could contribute to progression from MM to sPCL. Table +. The results from S100A-stimulated melanoma cells corroborate previous findings from our own laboratory in that DCA was selectively cytotoxic to melphalan-resistant myeloma cells with acquired Warburg metabolism [GBP2 (5.92-fold up-regulated in sPCL) is a member of the interferon-induced guanylate family of GTPases. It has been shown to induce proliferation in mouse fibroblasts  and to rtabolism . Finallytabolism .per se. The retinol-binding protein RBP1 (10.3-fold down-regulated in sPCL) is a tumor suppressor protein that is involved in the intracellular retinoic acid (RA) metabolism and treatment with RA inhibitors has been shown to inhibit MM cell growth and to induce apoptosis [The most down-regulated protein in sPCL was MARCKS (22.2-fold). MARCKS plays an important role in cell adhesion, spreading and invasion of various tumor cells , 78 and poptosis . Moreovepoptosis . The lowpoptosis . The clipoptosis  and prolpoptosis . Sheddinpoptosis . Notably6 deoxynucleosides in MM and sPCL, respectively). These results indicate that aberrant methylation is not a major cause of the significant shift in protein expression during progression to sPCL. Thus the increased expression in DNMT1 rather reflects increased proliferation at the sPCL stage, in agreement with previous studies in human cancers showing that DNMT1 is proliferation dependent [The relatively large number of differentially expressed proteins 795) in the malignant plasma cells from the MM and sPCL stages in the same patient was somewhat surprising and suggested the involvement of global epigenetic alterations. Moreover, among the differentially expressed proteins, the majority  were down-regulated. Aberrant methylation of cytosine residues in the promotor regions is associated with transcriptional silencing  and epig in the mependent .In summary, we present the first comprehensive protein profile highlighting differentially expressed proteins accompanying progression of MM to sPCL in a single patient. Many of these are amenable to targeting by small molecule inhibitors currently approved for clinical use. Further studies must be undertaken to verify whether our findings represent a common phenotype in the progression of MM to sPCL, or to what extent this holds true for subgroups harboring specific genetic alterations. In this respect, our super-SILAC library can be a valuable tool for other research groups conducting quantitative proteome profiling in MM and sPCL.http://iclac.org/databases/cross-contaminations).Six human multiple myeloma- and two human B-cell lymphoma cell lines were used to generate a heavy super-SILAC library for quantitative proteome profiling. The multiple myeloma cell lines IH-1 , OH-2 2, KJON an13C615N2-lysine  and 13C615N4-arginine   instead of the natural amino acids, dialyzed fetal bovine or human serum as appropriate, L-glutamine (100 μg/mL) and gentamicin (20 μg/mL). For IL-6 dependent cell lines  1 ng/mL recombinant human interleukin IL-6  was added. The Melphalan-resistant RPMI8226-LR5 was maintained under constant selection through the addition of 1 μM melphalan  twice weekly. Cells were cultured for at least eight passages in the SILAC medium at 37°C in a humidified atmosphere containing 5% CO2 in order to accomplish near complete labeling. Incorporation was examined by separate quantitative LC-MS/MS analysis and equal amounts of protein from each of the heavy lysates were premixed to generate the super-SILAC mix.All cell lines were passaged twice weekly in SILAC medium consisting of RPMI-1640  medium with + cells were stored in DMSO at -80°C in the Norwegian Myeloma Biobank at St. Olav's Hospital prior to analysis. The study was approved by the Regional committee for medical and health research ethics in Mid-Norway  and conforms to the Declaration of HelsinkiAt diagnosis her disease was characterized by Hgb 11.3 mg/dl, M protein of IgG-kappa type 20,5 g/l, 62% plasma cells in the bone marrow, multiple osteolytic lesions, ISS stage 2. t was detected whereas t, del 13 and del 17 were negative by FISH. During the initial four years she was sensitive to induction treatment with cyclophosphamide/bortezomib/dexamethasone followed by high dose melphalan with autologous stem cell transplantation , bortezomib/melphalan/prednisone  and cyclophosphamide/prednisone  and resistant to thalidomide/prednisone and lenalidomide/prednisone. After four years, plasma cell leukemia was diagnosed with 35% plasma cells in blood smears. She was then resistant to all drugs and died after two months. Malignant CD138+ cells were individually isolated and each subjected to four technical MS replicate analyses. At the time of sPCL diagnosis, six blood samples were collected over a time span of four days, each from which two aliquots of CD138+ cells were individually isolated and each subjected to two technical MS replicate analyses.A bone marrow sample was collected prior to the first treatment and blood samples were collected when sPCL had developed four years later. Plasma cells were purified by CD138+ magnetic-activated cell sorting (MACS) microbeads . For MM samples, two aliquots of CD138We employed methanol/chloroform  protein 3CN. Peptides were eluted with a linear gradient of 0-30% solvent B over 252 min, followed by 5 min at 100% B and 5 min at 100% A. Peptides were analyzed on the mass spectrometer operating in positive ion- and data dependent acquisition (DDA) mode using the following parameters: Electrospray voltage 2.2 kV, CID fragmentation with normalized collision energy 35, automatic gain control (AGC) target value of 1E6 for Orbitrap MS and 1E4 for MS/MS scans. Each MS scan (m/z 400–1600) was acquired at a resolution of 120,000 FWHM, followed by 20 MS/MS scans triggered for intensities above 500 and selected with an isolation window of 2 Th, at a maximum ion injection time of 200 ms for MS and 50 ms for MS/MS scans. Raw files were analyzed with MaxQuant v 1.5 [2 in order to have a better approximation to normal distribution of super-SILAC ratios. Further, the median of technical replicates were calculated to represent the samples in order to be less sensitive to outliers. These are presented in in Peptides were analyzed on a LC-MS/MS platform consisting of an Easy-nLC 1000 UHPLC system (Thermo Scientific/Proxeon) interfaced with an LTQ-Orbitrap Elite hybrid mass spectrometer (Thermo Scientific) via a nanospray ESI ion source . Peptides were injected onto a C-18 trap column  and further separated on a C-18 analytical column . The LC was operated at 250 nL/min over 262 min with solvent A consisting of 0.1% formic acid in water and solvent B of 0.1% formic acid in CHnt v 1.5  using itnt v 1.5 . Values The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium  via the


Quantifying differences or similarities in connectomes has been a challenge due to the immense complexity of global brain networks. Here we introduce a noninvasive method that uses diffusion MRI to characterize whole-brain white matter architecture as a single local connectome fingerprint that allows for a direct comparison between structural connectomes. In four independently acquired data sets with repeated scans , we show that the local connectome fingerprint is highly specific to an individual, allowing for an accurate self-versus-others classification that achieved 100% accuracy across 17,398 identification tests. The estimated classification error was approximately one thousand times smaller than fingerprints derived from diffusivity-based measures or region-to-region connectivity patterns for repeat scans acquired within 3 months. The local connectome fingerprint also revealed neuroplasticity within an individual reflected as a decreasing trend in self-similarity across time, whereas this change was not observed in the diffusivity measures. Moreover, the local connectome fingerprint can be used as a phenotypic marker, revealing 12.51% similarity between monozygotic twins, 5.14% between dizygotic twins, and 4.51% between none-twin siblings, relative to differences between unrelated subjects. This novel approach opens a new door for probing the influence of pathological, genetic, social, or environmental factors on the unique configuration of the human connectome. The local organization of white matter architecture is highly unique to individuals, making it a tangible metric of connectomic differences. The variability in local white matter architecture is found to be partially determined by genetic factors, but largely plastic across time. This approach opens a new door for probing the influence of pathological, genetic, social, or environmental factors on the unique configuration of the human connectome. PLOS Computational Biology Methods paper.This is a The specific brain characteristics that define an individual are encoded by the unique pattern of connections between the billions of neurons in the brain . This colocal connectome was proposed as an alternative measure to overcome the limitations of diffusion MRI fiber tracking [To this end, studies have used diffusion MRI (dMRI) to measure the architecture of white matter pathways using the diffusion properties of water molecules, 6. Thistracking –13. The n = 11, 25, 60, 118, see In this study, we used this local connectome feature vector as a fingerprint to quantify similarities and difference between two white matter architectures. To evaluate the performance of our approach, we used four independently collected dMRI datasets  with repeat scans for a subset of the subjects . The Euclidian difference  was used as a single difference estimate between any two fingerprints. For each dataset, we computed within-subject differences  and between-subject differences . To quantify how well the local connectome fingerprint captures between-subject difference, we used four independently collected dMRI datasets . For repeat scans acquired within 3 months, the probability of mistaking two samples of the same subject’s local connectome fingerprint as coming from two different individuals was low enough to consider the local connectome fingerprint a highly reliable measure of individual subject uniqueness.This within-subject consistency suggests that the local connectome fingerprint could be used as a unique subject identifier. To assess this, we used a linear discriminant analysis (LDA) classifier  to deter−4, 5.6×10−3, 6.9×10−3, and 7.2×10−3, for dataset I, II, III, and IV, respectively. The corpus callosum fingerprint itself already achieved more than 99% accuracy in subject identification. This suggests that the high individuality of the local connectome fingerprint is due to the microscopic characteristics of the white matter architecture.Since gross anatomical patterns such as gyral and sulcal folding can be highly specific to an individual, it is possible that the unique features we observed in the local connectome fingerprint reflected an artifact of the spatial normalization process. To evaluate this, we retested our uniqueness within a restricted white matter mask that only covered the median sagittal sections of the corpus callosum defined by the Johns Hopkins University white matter atlas . This “cDiffusivity-based metrics, such as FA, axial diffusivity (AD), and radial diffusivity (RD), also reveal microscopic structure of white matter systems. To compare these measures against SDF, we used the same analysis and replaced the SDF-based measures with FA, AD, and RD values of the corresponding voxels. Our analysis showed that the d-prime values of the FA-based fingerprint were 4.84, 4.70, 4.56, and 3.60, for dataset I, II, III, and IV, respectively. All values were substantially smaller than the local connectome fingerprint. The leave-one-out cross-validation analysis showed that classification error of the FA-based fingerprint was 0%, 0.18%, 0.22%, and 0.87%. While FA-based fingerprints also have high uniqueness with classification error less than 1%, the performance is not superior to the 0% leave-one-out cross-validation error achieved by the local connectome fingerprintWe also analyzed the performance of AD-based fingerprints, producing d-prime values of 4.20, 4.07, 4.33, and 3.68, for datasets I, II, III, and IV, respectively. The performance was similar to FA-based fingerprints and substantially lower than those of the local connectome fingerprint. The leave-one-out cross-validation analysis showed a classification error of 0.15% in dataset IV. While no misclassification was found in dataset I, II, III, the generalized extreme value distribution showed a classification error of 0.18%, 0.29%, and 0.18%, respectively. The AD-based fingerprint was also inferior to the local connectome fingerprint.−6 for the local connectome fingerprint and 0.28% for the RD-based fingerprint. By contrast, in dataset IV, which had the longest time interval (around a year), the classification errors were 5.5×10−3 for the local connectome fingerprint and 3.1×10−3 for the RD-based fingerprint. The uniqueness of the local connectome fingerprint dropped substantially over time. The possible causes may include biological causes, such as neuroplasticity, or more systematic error, such as any change in data acquisition due to software updates or scanner resonance changes. This issue was further investigated in the “Neuroplasticity revealed by the local connectome fingerprint” section below. To summarize, compared with diffusivity-based fingerprints, the local connectome fingerprints exhibited the greatest reliability for repeat scans acquired within 3 months.The analysis on RD showed a slightly different result. The d-prime values for RD were 7.87, 9.10, 8.79, and 5.80, which were substantially better than FA-based and AD-based fingerprints. Compared with the local connectome fingerprint, it is noteworthy that the local connectome fingerprint substantially outperformed RD for repeat scans within 3 months (14.84 and 12.80 versus 7.87 and 9.10), but not for repeat scans with a longer time interval . The classification error also showed a similar pattern. In dataset I, which had the shortest time interval (less than 16 days), the classification errors were 4.25×10We further compared the local connectome fingerprint with region-to-region connectivity estimates from diffusion MRI fiber tracking. The same analysis pipeline used for the local connectome fingerprint was used to calculate leave-one-out cross-validation error for the traditional connectivity matrix. The d-prime values for the region-to-region connectivity matrices in dataset I, II, III, and IV were at 3.44, 2.06, 2.41, and 2.25, respectively. The classification error for datasets I, II, III, and IV were 3.6%, 13.65%, 11.81%, and 9.48%, respectively . While the classification accuracy for the traditional connectivity matrices is still quite high and similar to what has previously been observed in resting state functional connectivity estimates , it is cOur analysis of within-subject differences hinted at the possibility of changes in the local connectome over time, and thus we further examined how time impacts the uniqueness of local connectome fingerprints. If the local connectome fingerprint is sensitive to neuroplasticity, a longer interval should result in decreased similarity between repeat scans of the same individual. To test this, we calculated the similarity of within- subject local connectome fingerprints as a percentage of the mean between-subject difference see . A similn = 24), which was acquired with the widest range of time interval between repeat scans (1~3 months). p = 0.0023). To further quantify the change of similarity in the local connectome fingerprint, we used linear regression to calculate the coefficient (slope) between the time interval and similarity. The results showed that the similarity dropped at a rate of 12.79% per 100 days. It is noteworthy that the identical analysis was applied to FA-based, AD-based, and RD-based fingerprints but none showed a significant trend .For this analysis, we calculated the similarity between repeat scans in dataset II . Post-hoc comparisons using Scheffé's S procedure showed (1) significantly higher similarity in MZ twins compared with all other groups , (2) significantly higher similarity in DZ twins compared with unrelated subjects (p = 0.001), and (3) significantly higher similarity in non-twin siblings compared with unrelated subjects (p = 0.0146). There was no significant difference between DZ twins and non-twin siblings (p = 0.9989).The local connectome fingerprint opens the possibility for comparing not only differences but also the similarities between individuals. To further illustrate how the local connectome fingerprint can be used to quantify white matter architecture as a phenotypic marker, we used a publicly available dMRI dataset of 486 subjects from Human Connectome Project , including 49 pairs of monozygotic (MZ) twins, 43 pairs of dizygotic twins (DZ) twins, and 96 pairs of non-twin siblings. While the local connectome fingerprints of MZ twins show generally similar patterns at the coarse level , there a2 statistics calculated from the Kruskal-Wallis test were smaller than the nonpermuted case (p< 0.0001). Thus the difference across groups was highly significant. Further permutation tests between pairs of subject groups were also conducted. The results showed (1) significantly higher similarity in MZ twins compared with DZ twins (p < 0.0001), non-twin siblings (p < 0.0001), and unrelated subjects (p < 0.0001), (2) significantly higher similarity in DZ twins compared with unrelated subjects (p < 0.0001), and (3) significantly higher similarity in non-twin siblings compared with unrelated subjects (p < 0.0001). There was no significant difference between DZ twins and non-twin siblings (p = 0.2697).To address the concern of data dependency, we conducted additional permutation tests to examine the difference in similarity across twins and unrelated subjects. A total of 10,000 permutations were calculated, and all χThis result is consistent with MZ twin sharing a higher genetic similarity, whereas DZ twins exhibit a similar genetic similarity on par with non-twin siblings.Local white matter architecture is so unique and highly conserved within an individual that it can be considered a unique neural phenotype. Here we show that this phenotype can be quantified by measuring the density of microscopic water diffusion along major white matter fascicles and producing a high dimensional vector that can be used to compute the distance between two structural connectomes, i.e., a local connectome fingerprint. The distance between two local connectome fingerprints reflects a low dimensional representation of both similarities and differences in whole-brain white matter pathways. Our analysis showed how the local connectome fingerprint exhibited unprecedentedly high between-subject distance, while generally low within-subject distances, allowing for it to be used as a reliable measure of the specific connective architecture of individual brains. This property paves the way for using the local connectome as a phenotypic marker of the structural connectome.The concept of local connectome is both conceptually and methodologically different from conventional connectomic measures. While most studies have emphasized on region-to-region connectivity  and igno−6 achieved by local connectome fingerprint.While any high dimensional representation of the human brain in a standard space has the potential to be used as fingerprint, we showed that the uniqueness of fingerprints generated from the local connectome was substantially higher than what was observed in diffusivity-based fingerprints as well as fingerprints derived from region-to-region connectivity reported by either dMRI or fMRI, as typically done in human connectomic studies , 20, 21.At first glance, it may seem possible that the high degree of uniqueness exhibited by the local connectome fingerprint could be due to variability in the spatial normalization process between individuals driven by the unique gyral or sulcal folding patterns in gray matter. While we still cannot rule out the effect of misalignment, our comparison with the FA-based fingerprints showed that the spatial normalization process does not fully contribute to the uniqueness observed in the local connectome fingerprint. Both FA-based fingerprints and the local connectome fingerprints used an identical spatial normalization mapping process, but the FA-based fingerprints had a much higher error rate in leave-one-out cross-validation (e.g. 0.87% for dataset IV) than the zero cross-validation error achieved by the local connectome fingerprint. Obviously, a substantial portion of the uniqueness was due to the microstructural white matter characteristics quantified in the SDF. Moreover, we observed favorable characterization of white matter uniqueness even when our analysis was restricted to a small portion of white matter with minimal influence of sulcal and gyral folding . These two findings support our claim that the local connectome fingerprint can reveal the unique characteristics of the white matter architecture. Finally, the between-subject differences are mostly located within the deep white matter at the central semiovale and the corpus callosum. This spatial specificity suggests that the uniqueness of the local connectome fingerprint is mostly driven by mesoscopic or microscopic architectural properties, not due to an artifact of unique folding geometry or the spatial normalization process.how fast water diffuses in tissue [how much water diffuses along the fiber pathways [It is important to point out that the local connectome fingerprint is based on a physical measurement that is different from diffusivity-based metrics such as FA, AD, and RD. To further compare their physical meanings, diffusivity quantifies n tissue  and is sn tissue , such asn tissue –26. Thispathways , 27 and pathways , 28, 29.The high degree of uniqueness in the local connectome within an individual can be used to reflect a quantifiable phenotype of neural organization. As illustrated in our analysis of twins, the similarity in monozygotic twins was around twice as much of the dizygotic twins, whereas our post-hoc analysis did not find significant similarity difference between dizygotic twins and non-twins siblings. These results are highly suggestive that genetics contribute a substantial portion to the overall construction of the local connectome, which is consistent with previous studies showing high heritability in cortical connections , 31 and It is important to point out that the highest similarity between repeat scans was around 70~80% in our study. This indicates that 20–30% of variability in the local connectome may arise from artifacts that decrease signal-to-noise ratio, such as cardiovascular and respiratory artifacts or computation error. This number reflects the limit of the local connectome fingerprint in detecting an anomaly in the individuals as well as finding differences in a group study. For example, we could not accurately identify whether two scans were from a twin pair because the similarity between twins was only around 12.51%. However, if a disease causes a white matter change with more than 30% difference in similarity, the local connectome fingerprint may be able to detect it. In a group study, increasing the number of subjects can average out the effect of noise and error on the similarity, allowing us to find a group difference that is substantially small. The similarity index from repeat scans allows us to gauge the strength and limitation of the local connectome fingerprint and prospectively, to develop a strategy to improve its performance.2. TR = 9916 ms, TE = 157 ms, voxel size = 2.4×2.4×2.4 mm, FoV = 231×231 mm.The first dataset included a total of 11 subjects . Each subject had three diffusion MRI scans within 16 days on a Siemens Trio 3T system at the University of California, Santa Barbara. All methods were approved by the local institutional review board at the University of California, Santa Barbara. The diffusion MRI was acquired using a twice-refocused spin-echo EPI sequence. A 257-direction full-sphere grid sampling scheme was used. The maximum b-value was 5000 s/mm2 (b-table available at http://dsi-studio.labsolver.org). TR = 9600 ms, TE = 130 ms, voxel size = 2.5×2.5×2.5 mm.The second set of data included a total of 24 subjects . All participants were scanned on a Siemens Tim Trio 3T system at National Taiwan University, and all subjects had their second scan at 1~3 months. All methods were approved by the local institutional review board at National Taiwan University. The diffusion MRI was also acquired using a twice-refocused spin-echo EPI sequence. The diffusion scheme is a 101-direction half-sphere grid sampling scheme with b-max = 4000 s/mm2. TR = 9916 ms, TE = 157 ms, voxel size = 2.4×2.4×2.4 mm, FoV = 231×231 mm.The third set of data included a total of 60 subjects . All participants were scanned on a Siemens Verio 3T system at Carnegie Mellon University, and 14 of the 60 subjects had their second scan at 6 months. All methods were approved by the local institutional review board at Carnegie Mellon University. The diffusion MRI was also acquired using a twice-refocused spin-echo EPI sequence. A 257-direction full-sphere grid sampling scheme was used. The maximum b-value was 5000 s/mm2. The in-plane resolution was 2.4 mm. The slice thickness was 2.4 mm.The fourth set of diffusion data included a total of 118 subjects  that were also scanned on a Siemens Verio 3T system at the Carnegie Mellon University. All methods were approved by the local institutional review board at the University of Pittsburgh and Carnegie Mellon University. 44 of them had another scan after one year. The diffusion images were acquired on a Siemens Verio scanner using a 2D EPI diffusion sequence. TE = 96 ms, and TR = 11100 ms. A total of 50 diffusion sampling directions were acquired. The b-value was 2000 s/mm2. The total number of diffusion sampling directions was 90, 90, and 90 for each of the shells in addition to 6 b0 images. The total scanning time was approximately 55 minutes. The scan data included 49 pairs of monozygotic twin, 43 pairs of dizygotic twins, and 96 pairs of siblings. We used the pre-processed data provided by the consortium in our analysis. Carnegie Mellon University Institutional Review Board (IRB) reviewed the research protocol for the data analysis in accordance with 45 CFR 46 and CMU’s Federal-wide Assurance. The research protocol has been given approval as Exempt by the IRB on March 12, 2014, in accordance with 45 CFR 46.101(b)(4) (IRB Protocol Number: HS14-139).The fifth dataset was from the Human Connectome Projects  acquired by Washington University in Saint Louis and University of Minnesota. The diffusion MRI data were acquired on a Siemens 3T Skyra scanner using a 2D spin-echo single-shot multiband EPI sequence with a multi-band factor of 3 and monopolar gradient pulse. A total of 486 subjects  received diffusion scans. The spatial resolution was 1.25 mm isotropic. TR = 5500 ms, TE = 89.50 ms. The b-values were 1000, 2000, and 3000 s/mmhttp://dsi-studio.labslover.org), an open-source diffusion MRI analysis tool for connectome analysis. The source code is publicly available on the same website. As shown in All five datasets were processed using an identical processing pipeline implemented in DSI Studio  in the sentricle . A σ of http://dsi-studio.labsolver.org), was used as a common SDF sampling framework to provide a consistent set of sampling directions A common axonal directions atlas, derived from the Human Connectome Project (HCP) dataset  classifier provided in MATLAB  was used, and for each dataset, the classification error was estimated using leave-one-out cross-validation. We also used a modeling method to calculate the classification error if the leave-one-out cross-validation did not yield any classification error. The histograms of the within-subject and between-subject differences were fitted by the generalized extreme value distribution using the maximum likelihood estimator (gevfit) provided in MATLAB. To consider the non-negativity of the distribution, the estimated To compare local connectome fingerprint with region-to-region connectivity matrix, deterministic fiber tracking was appl1/d0), where d1 was the difference between two fingerprints, and d0 was the expected value of the differences between unrelated subjects scanned by the same imaging protocol. The similarity between MZ twins, DZ twins, non-twin siblings, and repeated scans was calculated and compared. The Kruskal-Wallis test was applied to four groups  with independent samples. To further study the similarity between repeat scans, the similarity indices were tested against their scanning time intervals by the Mann-Kendall test to study the effect of time interval on the local connectome fingerprints.The similarity index between two local connectome fingerprints was calculated by 100%×(1-d


Erratum to: European Journal of Plastic SurgeryDOI 10.1007/s00238-017-1281-3The article Scarring of the C8-T1 roots with partial avulsion in situ in total obstetric brachial plexus palsy, written by Mohammad M. Al-Qattan & Amel A.F El-Sayed, was originally published electronically on the publisher’s internet portal (currently SpringerLink) on 17 February 2017 without open access.http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 24 May 2017 to © The Author(s) 2017 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (The original article was corrected.


The microbump (μ-bump) reliability of 3D integrated circuit (3D-IC) packaging must be enhanced, in consideration of the multi-chip assembly, during temperature cycling tests (TCT). This research proposes vehicle fabrications, experimental implements, and a nonlinear finite element analysis to systematically investigate the assembled packaging architecture that stacks four thin chips through the wafer level underfill (WLUF) process. The assembly of μ-bump interconnects by daisy chain design shows good quality. Results of both TCT data and the simulation indicate that μ-bumps with residual SnAg solders can reach more than 1200 fatigue life cycles. Moreover, several important design factors in the present 3D-IC package influence μ-bump reliability. Analytical results show that the μ-bump’s thermo-mechanical reliability can be improved by setting proper chip thickness, along with a WLUF that has a low elastic modulus and a small coefficient of thermal expansion. E), and the coefficient of thermal expansion (CTE) of WLUF, on the fatigue lifespan of μ-bumps through reliable simulation methodology.Given that chip-stacking packages, combined with either silicon-based or glass/ceramic-based interposers, are promising frameworks ,2,3,4,5 3Sn4 intermetallic compound (IMC). In addition, the intermediate zone of the μ-bump contained a 2.5-μm-thick lead-free solder remnant. An enlarged view of the μ-bump assembly is displayed in the scanning electron microscopy (SEM) images in 3Sn4 increased  were obtained using the least-squares method. Given the 96.5Sn3.5Ag lead solder, the constants of D and λ were 21.9 and 0.93, respectively, as per Equation (1) [f) for the lead-free solder could be restated further, as follows:Moreover, the conversion of the plastic shear strain extent, tion (1) . By rewrf) of the μ-bump was predicted. The incremental equivalent plastic strain was adopted for the Coffin-Manson relationship because the shrunken bumps constructed using FC technology [Once chnology  contribu3Sn4 IMC displayed a thickness of ~1 μm between the 5 μm thick lead-free solder and the 3.5 μm thick nickel layer. x-directional displacements of the nodes along the symmetry axis of the model were fixed. For the node at the bottom of this axis, all degrees of freedoms were set to prevent rigid body motion during the analysis. On the assumption that intrinsic stresses, resulting from packaging assembly, are totally eliminated, the analytical structure was considered stress-free at room temperature, and the thermal cycling load was subject to a temperature range of −55 °C to 125 °C. At least three cycles were performed to numerically converge the incremental averaged equivalent plastic strains in this FEA investigation.This research considers two types of chip-stacking 3D-IC packaging structures in the construction of a finite element model. The first type validated failure mode and estimated μ-bump reliability by mounting an initial layer of 50-μm-thick stacked chips. The second type predicted the fatigue lifetime of the μ-bump in the first two layers of the proposed package architecture with four stacked chips. As the second to the fourth stacked chips were bonded, identical WLUF procedures were applied sequentially. As a result, the layout of the entire structure was bi-axially symmetrical. The complexity of these components can generate critical problems in finite element modeling and subsequent computer time. Thus, a 2D simulated model was constructed along the cross-section by cutting the A′-A′ line  that was3Sn4 IMC occurred at an elevated temperature (125 °C) during the loadings of TCT. As compared with the thickness-dependent critical strength of Ni3Sn4 IMC (65~85 MPa) [3Sn4 IMC was lower than 40 MPa. Furthermore, the predicted location of the maximum value of equivalent plastic strain in the critical μ-bump was identical to the experimental result. The number of mean cycles to failure in the assembly of four 50-μm-thick chips was predicted to be 1324 when a stable 1.57% incremental equivalent strain was substituted into Equation (3), which agrees with the experimental data from TCT. This strain was obtained through a simulated analysis. Therefore, the proposed simulation methodology is highly reliable. Accordingly, the following section analyzes and discusses the effects of several geometric and material characteristics of WLUF on the μ-bump reliability of the 3D-IC package with four stacked chips.The experimental result of TCT indicated that the assembly of a one stacked chip takes 1250 cycles, at approximately 50% of the accumulated failure rate. In eight four-chip stacking structure specimens, more than 1200 cycles were obtained for the μ-bump fatigue lifespan using WLUF. The cross-sections of μ-bumps were examined, as shown in ~85 MPa) , the IMCIn conventional FC technology, the thermal strain on solder joints because of the CTE mismatch between silicon chips and printed circuit boards can be reduced significantly by filling the underfills among them. Shearing stress usually dominates the occurrences of fractures in FC bumps. Unlike the aforementioned situation, however, the stress/strain distribution and structural deformation of the framework of the present package with chip stacking can be determined, based on the complicated material characteristics of the components  given the identical characteristics used in the chips and in the assembled substrate. Thus, the warpage of an entire package under temperature cycling load must be investigated to further interpret the mechanical behavior of a critical μ-bump. The effects of stacked chips of different thicknesses  on bump reliability were parametrically determined during the assembly of four chips. The E effect of WLUF was systematically examined, over a range of 0.5 GPa to 10 GPa, against stacked chips with several thicknesses. As simulated in 4.4. μ-Bump Reliability Determined by the CTE of WLUFThe magnitude of thermal stress/strain induced in the package structure by TCT loading must be managed, because it typically dominates the failure causes and long-term reliability of μ-bumps. As an important design factor of WLUF, the effect of CTE on the present 3D-IC package with four-chip stacking was therefore analyzed. In this study, CTEs at 5, 10, 20, and 40 ppm/°C, were examined with the WLUF elastic modulus fixed at 5.6 GPa. E, and the CTE of WLUF were determined. The results show that flexibility can be enhanced by scaling chip thickness down to 100 μm. This high flexibility can, in turn, deteriorate the μ-bump reliability. Hence, WLUF should be characterized by a low E and a small CTE, based on the analysis results of this study. Furthermore, it is believed that the thermal compression approach of WLUF could overcome the filled void issue, while the pitch of microbumps is continually scaled down to the range of 10~20 μm.In this study, four 50-μm-thick chips were stacked using the proposed WLUF process. To determine the quality of the μ-bump connection, as well as the subsequent tolerance of warpage, induced during the WLUF thermal compression procedure, this study implemented an electrical check via the daisy chain method. The improvement in resistance was slight but acceptable. In addition, the Coffin-Manson relationship, associated with lead-free solders, was combined with non-linear FEA to estimate the μ-bump reliability. As per the validation of TCT experimental data, under temperatures ranging from −55 °C and 125 °C, the proposed simulation methodology was highly reliable. The four 50-μm-thick chip packages exceeded 1200 cycles. The FEA-predicted occurrence of fracture at the region near the IMC/lead-free solder interface of the critical μ-bump was consistent with that obtained in actual testing. Based on the comparisons, several geometric and material property effects, including stacked chip thickness,


Flexible and efficient decision-making in complex environments can be achieved through constant interactions between the goal-directed and habitual systems. While goal-directed behavior is considered dependent upon Response-Outcome (R-O) associations, habits instead rely on Stimulus-Response (S-R) associations. However, the stimuli that support the S-R association underlying habitual responding in typical instrumental procedures are poorly defined. To resolve this issue, we designed a discrete-trials procedure, in which rats must wait for lever insertion and complete a sequence of five lever presses to obtain a reward (20% sucrose or grain-based pellets). Lever insertion thus constituted an audio-visual stimulus signaling the opportunity for reward. Using sensory-specific satiety-induced devaluation, we found that rats trained with grain-based pellets remained sensitive to outcome devaluation over the course of training with this procedure whereas rats trained with a solution of 20% sucrose rapidly developed habit, and that insensitivity to outcome devaluation in rats trained with sucrose did not result from a bias in general satiety. Importantly, although rats trained with pellets were sensitive to satiety-induced devaluation, their performance was not affected by degradation of instrumental contingency and devaluation by conditioned taste aversion (CTA), suggesting that these rats may also have developed habitual responding. To test whether the discrete-trials procedure biases subjects towards habitual responding, we compared discrete-trials to free-running instrumental responding, and found that rats trained with sucrose in a fixed-ratio 5 (FR5) procedure with continuous presentation of the lever were goal-directed. Together, these results demonstrate that discrete presentations of a stimulus predictive of reward availability promoted the formation of S-R habit in rats trained with liquid sucrose. Further research is necessary to explain inconsistencies in sensitivity to outcome devaluation when rats are trained with grain-based pellets. Flexible and efficient decision-making in complex environments can be achieved through constant interactions between the goal-directed and habitual systems. While a goal-directed system allows for the consideration of actions’ consequences, and flexible adaptation when those consequences change, habits are automatically triggered by antecedent predictive stimuli and do not require the anticipation of the outcome of each available action. In associative learning theories, goal-directed behavior has been defined as dependent upon Response-Outcome (R-O) associations, the response being mediated by the expectation of the outcome . Since goal-directed responding relies on the causal relationship between the response and the outcome, and the current motivational value of that outcome, the performance decreases when the instrumental contingency is degraded or when the value of the outcome is reduced  schedules have been shown to promote goal-directed behavior whereas overtraining or random-interval (RI) schedules promote the formation of habits Adams, . DickinsThe demonstration of habit as a positive result, i.e., the reliance on an S-R association, is challenging because the stimuli supporting the S-R association, and thus habitual performance, are poorly defined in instrumental procedures. These stimuli are situational cues being associated with the instrumental response through repeated reinforcement. As previously highlighted, the incidental nature of this association make the stimuli difficult to identify and manipulate . The protocol was approved by the institutional animal care and use committee of Johns Hopkins University.A total of 73 experimentally naive Long–Evans rats  weighing on average 300–330 g  and 220–240 g  at the start of training were individually housed and maintained in a light-  and temperature-controlled vivarium (21°C) with partial enrichment. All experiments were performed during the light cycle. Rats were maintained at 90% of their free-feeding weight and food rations were given 1–2 h after daily behavioral sessions. Water was available n = 4) or whose performance fell below two completed ratios for three consecutive sessions before a test (n = 2), were excluded.Training and testing occurred in conditioning chambers housed within sound-attenuating boxes . Rats underwent a single 30-min magazine training session, in which a reward  was delivered under a variable time-60 s schedule into a recessed magazine in the middle of the right wall of the chamber. Rats were next trained to press the left lever to earn a small aliquot of sucrose 20% (0.1 mL delivered over 3 s) or a single 45 mg grain-based pellet (Bioserv Biotechnologies), delivered in the adjacent magazine. Both rewards were delivered in an identical oval tray mounted in the magazine. Sessions ended after rats had earned a maximum of 30 rewards or 1 h had elapsed. The house-light, located on the ceiling of the operant chamber, remained illuminated during the full length of the session. After five sessions of this continuous reinforcement schedule (CRF), the discrete-trials procedure was introduced. Each session comprised 30 trials separated by 1-min inter-trial intervals. Every trial started with the insertion of the left lever. For the first two sessions, one lever press simultaneously resulted in the retraction of the lever, reward delivery, and the initiation of a new inter-trial interval  1; DT1). The response ratio was then increased to five . Failure to complete the ratio within 1 min was considered as an omission and resulted in lever retraction and initiation of a new inter-trial interval. Sensitivity to outcome devaluation was tested using sensory-specific satiety after various lengths of training. Animals failing to learn instrumental responding after five sessions of CRF , while the other half received the control  reward . Pre-feeding occurred for 1 h in feeding cages in the experimental room. Immediately after pre-feeding, rats were placed in the operant chambers for a 10-trial extinction session. The procedure was identical to that of training except that no reward was delivered. As in training, the lever extended at the start of each trial and retracted after 1 min or at the completion of the ratio. On the second test session, animals were pre-fed with the alternative reward prior to a second 10-trial extinction test.p = 0.5. To assess individual sensitivity to contingency degradation, we compared instrumental responding in the “pre-test” and “post-degradation” extinction sessions.One day prior to exposure to the contingency degradation procedure, rats trained to respond for sucrose or pellets on the DT5 schedule underwent a 10-trial extinction test to assess baseline reward seeking (“pre-test”). The next 3 days, rats underwent contingency-degradation training, followed by a final 10-trial extinction test to assess post-degradation reward seeking. Contingency-degradation sessions consisted of 10 reinforced trials and a free reward delivery during each inter-trial interval (variable time-30 s). Thus, the probability of receiving a reward after the completion of a five lever presses sequence decreased to Rats were assigned to “paired” and “unpaired” groups for taste aversion training such that baseline response rate, and previous sensitivity to outcome devaluation, were balanced across the two groups, for each outcome type. Conditioned taste aversion (CTA) was induced in the animals’ home cages over two cycles of 2 days. On the first day of each cycle, rats in the paired group received 10 min access to their training reward whereas rats in the unpaired group received nothing. Immediately after this 10 min period, all rats (both paired and unpaired subjects) received an intraperitoneal injection of a 0.3 M lithium chloride (LiCl) solution (6 mL/kg). On the second day of each cycle, rats in the unpaired group received 10 min access to their training reward but none of the rats received an injection. Thus, exposure to the reward and LiCl injections was identical for the two groups but only rats from the paired group were subjected to LiCl-induced illness paired with reward consumption. To confirm the induction of CTA for paired rats and assess the level of generalization from the home cage to the experimental chamber, rats were exposed to their training reward in the magazine of the experimental chamber for 5 min. The magazine food cup was filled with 3 mL of sucrose or 25 grain-based pellets. On the test day, rats were then placed in their experimental chambers for a 10-trial extinction session identical to that described above.Lever presses and port entries were collected throughout training and test. In the DT5 procedure, mean response rate was expressed in responses per second and was measured as the number of lever presses per trial divided by the time of lever availability, averaged across trials. Time of lever availability was set to 60 s in omission or incomplete trials. To compare change in sucrose and pellets consumption during taste aversion learning in Experiment 4, consumption in the home cage was expressed as the percentage of the amount eaten prior to treatment. In the operant box, consumption was expressed as the percentage of presented food that was consumed.post hoc comparisons when indicated, using Tukey’s Honest Significant Difference (HSD) test. Significance was assessed against a type I error rate of 0.05.Four female rats were used in the last experiment. There were no main effects of sex or devaluation by sex interactions in this experiment. Analyses were thus conducted on pooled data. Notably, when data of males only were examined, the findings remained the same. Note that the small sample size of female rats precludes any strong conclusions regarding sex differences in habit formation. All data were subjected to repeated measures analyses of variance, followed by n = 6) or grain-based pellets (n = 7) underwent satiety-based outcome devaluation after one or six sessions of DT5 training. After pre-training , rats rapidly learned to press the lever for sucrose or pellets in the DT5 procedure, with the number of lever presses near the maximum possible from the first DT5 session  = 17.14, p < 0.0001), but did not differ based on the training reward  = 0.60, ns). Sensitivity to satiety-induced devaluation of the outcome was tested after one and six DT5 training sessions, using 20% maltodextrin and 20% sucrose as control rewards  for rats trained with 20% sucrose and grain-based pellets, respectively. After 1 training session, rats were sensitive to outcome devaluation, as indicated by a main effect of devaluation on lever pressing  = 8.59, p < 0.05) and on normalized response rate  = 22.94, p < 0.001). After five additional training sessions, responding was no longer significantly affected by outcome devaluation, as indicated by similar number of lever presses  = 4.13, ns) and similar normalized response rate  = 2.74, ns) in the valued and devalued conditions. Although there were no effects of reward nor reward by devaluation interactions, rats trained with pellets seemed more sensitive to satiety-induced devaluation than rats trained with sucrose, particularly after six training sessions.To assess the presence of sensitivity to outcome devaluation after training in a “discrete trials” (DT) procedure, designed such that each trial is initiated by lever insertion, rats trained to respond for a 20% solution of sucrose  and grain-based pellets (n = 5), respectively. Despite similar performance during training, both in term of lever presses  = 0.0857, ns), sensitivity to satiety-induced devaluation differed as a function of the training reward  = 33.59, p < 0.001, normalized response rate F = 49.27, p < 0.001). However, this devaluation effect interacted with the training reward  = 9.41, p < 0.05, normalized response rate F = 10.49, p < 0.05). Post hoc analyses of these interactions revealed that lever pressing and normalized response rate for pellets remained sensitive to devaluation across tests  whereas lever pressing and normalized response rate for sucrose were not affected by outcome devaluation  = 62.39, p < 0.01, test 2: F = 22.94, p < 0.01, test 3: F = 11.79, p < 0.05) but not for animals trained with sucrose . There was a significant effect of test  = 8.18, p < 0.01, normalized response rate, F = 22.02, p < 0.0001) but no devaluation by test , reward by test  or devaluation by reward by test  interactions.To explore possible differences in outcome sensitivity in sucrose- and pellet-trained rats, an additional experiment with a longer time course was conducted. In devaluation tests of the second experiment, grain-based pellets and sucrose 20% were used as the alternative control rewards for rats trained with sucrose 20%  = 22.08, p < 0.01, 17d: F = 12.82, p < 0.01, 43d: F = 24.73, p < 0.01) and normalized response rate  = 16.52, p < 0.01, 17d: F = 18.59, p < 0.01, 43d: F = 16.86, p < 0.01). This devaluation effect interacted with the training reward on lever pressing at 17d and 43d  = 6.42, p < 0.05, 43d: F = 7.78, p < 0.05) but not at 5d  = 2.34, ns). The devaluation by training reward interaction was only significant at 43d considering normalized response rate  = 6.65, p < 0.05). Post hoc analyses of each interaction revealed a sensitivity to outcome devaluation for rats trained with pellets, but not for rats trained with sucrose.To assess the time course of the development of insensitivity to satiety-induced devaluation in rats trained with sucrose, a repeated measure ANOVA was conducted on each test separately. This analysis revealed a main effect of devaluation on tests 5d, 17d and 43d for lever pressing  = 71.48, p < 0.0001), the unbalanced consumption of these rewards during the test may create a bias in general satiety and explain the dissociation observed in the results depicted in Figure These results suggest that rats trained with sucrose 20% progressively developed habitual responding while rats trained with pellets remained goal-directed. Because caloric intake was higher during pre-feeding with pellets compared to sucrose in this experiment  was trained with sucrose 20% and was pre-fed with sucrose 20% or an isocaloric unsweetened solution of maltodextrin 20% (polycose) as the control reward during the devaluation test (Experiment 3). As expected, rats successfully learned the DT5 task, as indicated by their high level of lever pressing and progressive increase in response rate across sessions  = 29.57, p < 0.0001). Sensitivity to outcome devaluation was tested after 5 and 15 DT5 sessions. Caloric intake during pre-feeding access was similar in the valued and devalued conditions for both tests . Lever pressing and normalized response rates were not reduced in the devalued condition compared to the valued condition  = 5.09, p < 0.05) but no devaluation by test interaction .Therefore, to control for any bias in general satiety during pre-feeding, a new cohort of rats  or 20% sucrose (n = 12) was tested for sensitivity to satiety-induced devaluation, contingency degradation and devaluation induced by CTA. As expected, learning of the DT5 task was associated with high levels of responding  = 33.54, p < 0.0001) with no difference between groups  = 1.32, ns). Sensitivity to satiety-induced devaluation was tested after five DT5 sessions, using the alternative reward as control reward during pre-feeding in the valued condition . As expected from the first two experiments, sensitivity to outcome devaluation interacted with the training reward  = 7.62, p < 0.05, Figure F = 8.41, p < 0.01). Post hoc analyses of these interactions revealed that lever pressing and normalized response rates were significantly lower in the devalued condition compared to the valued condition for rats trained with pellets . In contrast, these measures did not differ for rats trained with sucrose (p > 0.05).To further investigate the differential sensitivity to outcome devaluation observed in the previous experiments, a new group of rats trained with grain-based pellets , could reduce hunger to a larger extent than pre-feeding with sucrose in the valued condition, creating an imbalance in motivational state during the test. To assess the response strategy of rats trained with pellets or sucrose in absence of bias related to pre-feeding conditions, this same group of rats was tested for degradation of contingency and devaluation by CTA.F values <2.1, ns). After two additional days of training, a CTA was induced. Pellets and sucrose were successfully devalued in paired rats compared to unpaired rats  = 26.36, P < 0.0001) without effect of reward, nor a devaluation by reward interaction . Importantly, the CTA readily generalized from the home-cage, where it was established, to the operant box, as indicated by a main effect of devaluation on sucrose and pellet consumption in the operant box  = 77.92, p < 0.0001). In this consumption test on day 3, a devaluation by reward interaction  = 9.39, p < 0.01) indicates that the sucrose solution was devalued to a larger extent than grain-based pellets . Despite devaluation of each training reward, paired rats did not show any reduction in lever presses . There was a significant effect of reward on lever pressing  = 5.33, p < 0.05) but no other significant main effects or interactions . Importantly, the insensitivity of pellet rats to devaluation by CTA cannot be explained by partial devaluation of grain-based pellets or additional training in the DT5 procedure between each test. Indeed, these results were replicated in another cohort of rats trained with pellets for five DT5 sessions. Despite the strong devaluation of their reinforcer, paired rats made similar numbers of lever presses with a similar response rate as unpaired rats , thus replicating results from this experiment.Rats were tested in the contingency degradation procedure after 1 additional week of training. This procedure did not affect response rate, neither for rats trained with pellets nor for rats trained with sucrose Figure . Comparis Figure . A two-fs Figure , or norms Figure , comparen = 10) was trained with 20% sucrose under an FR5 schedule of reinforcement with continuous access to the lever (Experiment 5). Lever pressing and response rate progressively increased across sessions  = 1.52, p < 0.05, Figure F = 2.34, p < 0.0001). Sensitivity to satiety-induced devaluation was tested after 5, 22 and 43 FR5 sessions. Grain-based pellets were used as the alternative (control) reward during the test to mimic the devaluation conditions of the second experiment. At each of the 3 tests, rats succeeded in modifying their responding according to the new outcome value, as indicated by a significant effect of devaluation on lever pressing  = 14.28, p < 0.01) and normalized response rate  = 20.55, p < 0.01). There was no main effect of test or a devaluation by test interaction .While the discrepancies in the results for rats trained with pellets raise important questions, the two devaluation procedures as well as the contingency degradation procedure suggest that rats trained with sucrose 20% in the DT5 procedure rapidly developed habitual responding. To test whether this insensitivity to outcome devaluation results from the structure of the DT5 procedure, an additional group of rats . Within-sequence port entries also decreased in the FR5 procedure, but remained significantly above zero  and higher compared to the DT5 procedure  = 65.99, p < 0.0001, main effect of sessions F = 11.62, p < 0.0001). In addition, performance in the DT5 procedure was numerically greater and less variable than performance in the FR5 procedure  = 38.87, p < 0.0001) and on the coefficient of variation of within-sequence response rate  = 28.41, p < 0.001), respectively. Response rates increased to a larger extent over training in the DT5 procedure compared to the FR5 procedure  = 8.49, p < 0.001). Finally, the 1st lever press latency was systematically shorter in the DT5 procedure  = 6.86, p < 0.05).The results above indicate that rats trained with sucrose in the FR5 procedure remained goal-directed whereas rats trained with the same reward in the DT5 procedure developed habit. To explore differences between these procedures we compared the microstructure of behavior and different measures of automaticity  at different points in training in Experiments 2 and 5. This analysis reveals that performance was more efficient in the DT5 procedure than in the FR5 procedure Figures . On the  Figures , as indiThe objective of this study was to determine whether the presence of a salient stimulus predictive of reward availability could promote the formation or expression of S-R habit. Our results demonstrate that providing this salient stimulus in the DT5 procedure promoted habitual responding in rats trained with 20% sucrose, as indicated by their insensitivity to outcome devaluation. Interestingly, rats trained with grain-based pellets remained persistently sensitive to satiety-induced devaluation, even after 8 weeks of DT5 training. This persistent sensitivity to outcome devaluation could result from a motivational bias during pre-feeding since rats trained with pellets were insensitive to contingency degradation and outcome-devaluation by CTA, suggesting that their behavior was in fact habitual. However, when sucrose was the reinforcer, the training schedule dictated whether responding was habitual or not. When rats were trained under the discrete-trials procedure, responding was insensitive to devaluation, and response measures were congruent with the development of a well-learned, automatic behavior. In contrast, when rats were trained under the more typical free-running fixed-ratio schedule, responding was sensitive to outcome devaluation, and response measures showed relatively greater trial-by-trial variability, congruent with a goal-directed behavior.A previous study demonstrated that satiety-induced devaluation test results can vary depending on the ability of the reinforcer to induce satiety  and goal-directed  and degradation of contingency, as well as through measures of behavioral automaticity. Although further experiments are necessary to explain inconsistencies in sensitivity to outcome devaluation for rats trained with pellets, these results demonstrate the strong influence of discrete reward-predictive cues on the control of instrumental performance. We propose, therefore, that a DT approach, in which the stimulus is readily identifiable and stimulus-triggered behavior can be quantified in detail, may be useful for probing the dependence of habits on the formation of S-R associations, and may ultimately allow for better definition of the place of habit in substance use disorders (Vandaele and Janak, YV designed and carried out experiments, analyzed data and wrote the manuscript. HJP carried out experiments and participated in data extraction and analysis. PHJ designed experiments and wrote the manuscript. All authors critically reviewed content and approved the final version for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.